The present invention relates generally to cytokine receptors, and more specifically, to leukemia inhibitory factor receptors.
Leukemia inhibitory factor (LIF) is a polypeptide hormone which plays a central role in the regulation of diverse adult and embryonic systems. LIF acts on a variety of cell types and has multiple biological activities. The diversity in biological activity is reflected in the various synonyms of LIF, which include hepatocyte stimulating factor III (HSF III); Baumann and Wong, J. Immunol. 143:1163, 1989); cholinergic nerve differentiation factor (CNDF; Yamamori et al., Science 246:1412, 1990); melanoma-derived lipoprotein lipase inhibitor (MLPLI; Mori et al., Biochem. Biophys Res. Comm. 160:1085, 1989); human interleukin for DA cells (HILDA; Moreau et al., Nature 336:690, 1988); differentiation factor (D-factor; Tomida et al., J. Biol. Chem. 259:10978, 1984); differentiation inhibitory factor (DIF; Abe et al., J. Biol. Chem. 264:8941, 1989); differentiation inhibitory activity (DIA; Smith and Hooper, Devel. Biol. 121:1, 1987); and differentiation retarding factor (DRF; Koopman and Cotton, Exp. Cell. Res. 154:233, 1984).
The diversity of biological activities ascribed to LIF, whether differentiation inhibition or stimulation, proliferation or functional activation, is mediated by specific plasma membrane receptors which bind LIF. Despite the wide range of biological activities mediated by LIF, it is believed that LIF receptors (LIF-R) are highly conserved in a variety of species and expressed on a large variety of cells, since the ligand is highly conserved between species (Gough et al., Proc. Natl. Acad. Sci USA 85:2623, 1988; Yamamori et al., Science 246:1412, 1990). LIF receptors have been identified by ligand affinity cross-linking techniques on various cell lines, including monocyte-macrophages (Hilton, et al., Proc. Natl. Acad. Sci. USA 85:5971, 1988), and also on some non-hematopoietic cells including osteoblasts, placental trophoblasts, and liver parenchymal cells (Metcalf et al., Blood 76:50, 1990). Such studies indicate that LIF-R has a molecular weight of 90 kDa (Jacques et al., 5th Symposium sur les Marqueurs de l'inflammation, Lyon Sep. 25-27, 1990, Abstract No. 37, page 122 (bioMerieux sa, Lyon, France). Characterization of LIF receptors by Scatchard analysis of binding isotherms has demonstrated that specific cell surface receptor molecules from a variety of target cells have approximately the same affinity for LIF (40-100 pM) and are present in low numbers (150 to 2,500 receptors per cell) on all cells types tested.
In order to study the structural and biological characteristics of LIF-R and the role played by LIF-R in the responses of various cell populations to LIF stimulation, or to use LIF-R effectively in therapy, diagnosis, or assay, homogeneous compositions are needed. Such compositions are theoretically available via purification of receptors expressed by cultured cells, or by cloning and expression of genes encoding the receptors. Prior to the present invention, however, several obstacles prevented these goals from being achieved.
First, although some cell lines have been identified which express LIF-R, such cell lines express LIF-R only in very low numbers (150 to 2,500 receptors/cell), which has impeded efforts to purify receptors in amounts sufficient for obtaining amino acid sequence information or generating monoclonal antibodies. The low numbers of receptors has also precluded any practical translation assay-based method of cloning.
Second, even if LIF-R protein compositions of sufficient purity could be obtained to permit N-terminal protein sequencing, the degeneracy of the genetic code may not permit one to define a suitable probe without considerable additional experimentation. Many iterative attempts may be required to define a probe having the requisite specificity to identify a hybridizing sequence in a cDNA library. Although direct expression cloning techniques avoid the need for repetitive screening using different probes of unknown specificity and have been useful in cloning other receptors (e.g., IL-1R), they have not been shown to be sufficiently sensitive to identify LIF-R clones from cDNA libraries derived from cells expressing low numbers of LIF-R.
Thus, efforts to purify the LIF-R or to clone or express genes encoding LIF-R have been impeded by lack of purified receptor or a suitable source of receptor mRNA.